Organotypic Culture of Full-thickness Adult Porcine Retina

Summary

Here we describe a cost-effective technique for organotypic culture of adult porcine retina for seven days. Briefly, a sterile filter paper was used to lift the neural retina off from the RPE and place photoreceptor side up on an insert raised by a custom-made stand.

Abstract

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human retina and is therefore a valuable species for studying mechanisms of human retinal injury and degenerative disease. Here we describe a cost-effective technique for organotypic culture of adult porcine retina isolated from eyes obtained from an abattoir. After removing the anterior segment, a trephine blade was used to create multiple neural retina-Bruch's membrane-RPE-choroid-sclera explants from the posterior segment of adult porcine eyes. A piece of sterile filter paper was used to lift the neural retina off from each explant. The filter paper-retina complex was cultured (photoreceptor side up) atop an insert, which was held away from the bottom of the culture dish by a custom-made stand. The stand allows for good circulation of the culture medium to both sides of the retina. Overall, this procedure is simple, reproducible, and permits preservation of native retinal structure for at least seven days, making it a useful model for a variety of morphological, pharmacological, and biochemical studies on mammalian retina.

Protocol

1. Making a custom-made stand for cell culture inserts

The base of a 5 ml pipette tip (ISC BioExpress, #P-3250-19) has an inner diameter of 13 mm, which perfectly matches the bottom size (12.6 mm, outer diameter) of a 10 mm (inner diameter) NUNC cell culture insert (8 μm, Polycarbonate membrane, Thermal, #137443). To make the stand, the pipette was cut off near the base to produce a 6 mm long hollow cylinder. Then, pieces were removed from the side of the cylinder using a flame-heated blade to create 3 legs (4 mm in height) under a ring (2 mm in height), which raised the height of the inserts by approximately 5 mm.

Discussion

Vision researchers have established a variety of retinal culture systems, including organotypic systems, to study a variety of issues, such as retinal stem cell transplantation¹, retinal regeneration², and exogenous gene expression^3-5. However, adult mammalian retina is difficult to maintain in vitro, mainly due to the high-energy demands of the photoreceptors⁶. Koizumi et al. described a rabbit retinal organotypic culture system in which the oxygen supply for.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
MEM	Invitrogen	10370-021
5 mL pipette tip	ISC BioExpress	P-3250-19
NUNC cell culture insert	Thermal Scientific, Inc.	137443	8μm, Polycarbonate
Whatman 4M Filter paper	Whatman, GE Healthcare	1004
DMEM	Cellgro	10-013-CV
Agar	Fluka	05040
Fungizone Amphotericin B	Invitrogen	15290018
Trephine blades	Bausch and Lomb	T3096	6.00mm
O.C.T. Compound	Electron Microscopy Sciences	62550-01	4583
Cryostat	Leica Microsystems	CM1900
Vibratome	Leica Microsystems	Series 1000
Confocal microscopy;	Carl Zeiss, Inc.	LSM510
Anti-Synaptic Vesicle Protein 2 (SV2) mouse monoclonal antibody	DSHB	N/A
Anti-Glial Fibrillary Acidic Protein (GFAP) polyclonal rabbit antibody	Dako	DAKO
Propidium Iodide (PI)	Sigma-Aldrich	P4170